Purification, Identification, and Characterization of a Glycoside Hydrolase Family 11-Xylanase with High Activity from Aspergillus niger VTCC 017.

Xylanases (EC 3.2.1.8) have been considered as a potential green solution for the sustainable development of a wide range of industries including pulp and paper, food and beverages, animal feed, pharmaceuticals, and biofuels because they are the key enzymes that degrade the xylosidic linkages of xylan, the major component of the second most abundant raw material worldwide. Therefore, there is a critical need for the industrialized xylanases which must have high specific activity, be tolerant to organic solvent or detergent and be active during a wide range of conditions, such as high temperature and pH. In this study, an extracellular xylanase was purified from the culture broth of Aspergillus niger VTCC 017 for primary structure determination and properties characterization. The successive steps of purification comprised centrifugation, Sephadex G-100 filtration, and DEAE-Sephadex chromatography. The purified xylanase (specific activity reached 6596.79 UI/mg protein) was a monomer with a molecular weight of 37 kDa estimating from SDS electrophoresis. The results of LC/MS suggested that the purified protein is indeed an endo-1,4-ß-D-xylanase. The purified xylanase showed the optimal temperature of 55 °C, and pH 6.5 with a stable xylanolytic activity within the temperature range of 45-50 °C, and within the pH range of 5.0-8.0. Most divalent metal cations including Zn2+, Fe2+, Mg2+, Cu2+, Mn2+ showed some inhibition of xylanase activity while the monovalent metal cations such as K+ and Ag+ exhibited slight stimulating effects on the enzyme activity. The introduction of 10-30% different organic solvents (n-butanol, acetone, isopropanol) and several detergents (Triton X-100, Tween 20, and SDS) slightly reduced the enzyme activity. Moreover, the purified xylanase seemed to be tolerant to methanol and ethanol and was even stimulated by Tween 80. Overall, with these distinctive properties, the putative xylanase could be a successful candidate for numerous industrial uses.

Method for the Production and Purification of Plant Immuno-Active Xylanase from Trichoderma.

Plants lack a circulating adaptive immune system to protect themselves against pathogens. Therefore, they have evolved an innate immune system based upon complicated and efficient defense mechanisms, either constitutive or inducible. Plant defense responses are triggered by elicitors such as microbe-associated molecular patterns (MAMPs). These components are recognized by pattern recognition receptors (PRRs) which include plant cell surface receptors. Upon recognition, PRRs trigger pattern-triggered immunity (PTI). Ethylene Inducing Xylanase (EIX) is a fungal MAMP protein from the plant-growth-promoting fungi (PGPF)-Trichoderma. It elicits plant defense responses in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum), making it an excellent tool in the studies of plant immunity. Xylanases such as EIX are hydrolytic enzymes that act on xylan in hemicellulose. There are two types of xylanases: the endo-1, 4-ß-xylanases that hydrolyze within the xylan structure, and the ß-d-xylosidases that hydrolyze the ends of the xylan chain. Xylanases are mainly synthesized by fungi and bacteria. Filamentous fungi produce xylanases in high amounts and secrete them in liquid cultures, making them an ideal system for xylanase purification. Here, we describe a method for cost- and yield-effective xylanase production from Trichoderma using wheat bran as a growth substrate. Xylanase produced by this method possessed xylanase activity and immunogenic activity, effectively inducing a hypersensitive response, ethylene biosynthesis, and ROS burst.

Enterocin M-Producing Enterococcus faecium CCM 8558 Demonstrating Probiotic Properties in Horses.

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

Identification and characterization of α-xylosidase involved in xyloglucan degradation in Aspergillus oryzae.

Aspergillus oryzae produces hydrolases involved in xyloglucan degradation and induces the expression of genes encoding xyloglucan oligosaccharide hydrolases in the presence of xyloglucan oligosaccharides. A gene encoding α-xylosidase (termed AxyA), which is induced in the presence of xyloglucan oligosaccharides, is identified and expressed in Pichia pastoris. AxyA is a member of the glycoside hydrolase family 31 (GH31). AxyA hydrolyzes isoprimeverose (α-D-xylopyranosyl-(1→6)-D-glucopyranose) into D-xylose and D-glucose and shows hydrolytic activity with other xyloglucan oligosaccharides such as XXXG (heptasaccharide, Glc4Xyl3) and XLLG (nonasaccharide, Glc4Xyl3Gal2). Isoprimeverose is a preferred AxyA substrate over other xyloglucan oligosaccharides. In the hydrolysis of XXXG, AxyA releases one molecule of D-xylose from one molecule of XXXG to yield GXXG (hexasaccharide, Glc4Xyl2). AxyA does not contain a signal peptide for secretion and remains within the cell. The intracellular localization of AxyA may help determine the order of hydrolases acting on xyloglucan oligosaccharides.

Characterization of a novel multi-domain xylanase from Clostridium clariflavum with application in hydrolysis of corn cobs.

OBJECTIVES: To develop a novel multi-catalytic domain (CD) xylanase Xyn2083 from Clostridium clariflavum by expression of its truncated forms in Escherichia coli and cooperation of xylanase with cellulase in the hydrolysis of waste lignocellulosic resources. RESULTS: Xyn2083 has two glycoside hydrolase family (GH) domains GH11 and GH10. These two catalytic domains functioned synergistically in xylan hydrolysis. The recombinant protein with GH11 domain, Xyn2083GH11, had the highest xylanase activity among three constructed truncated forms. The deletion of N-terminal extra amino acid residues of Xyn2083GH11 decreased catalytic activity as well as the stability of the enzyme. The hydrolysis rates of cellulose and xylan in the pretreated corn cobs were 90.56% and 72.80% with the addition of Xyn2083GH11 and cellulase, whereas those were 67.95% and 34.45% using sole cellulase respectively. The structural analysis of substrates indicated that the addition of Xyn2083GH11 led to a looser structure and more exposure of crystal cellulose for cellulase to approach. CONCLUSIONS: Since the native multi-CDs' xylanases are rare, the thermostable Xyn2083 provides a good source for functional studies of two CDs coexisted in one xylanase and for potential applications after modification.

Functional exploration of Pseudoalteromonas atlantica as a source of hemicellulose-active enzymes: Evidence for a GH8 xylanase with unusual mode of action.

To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a ß-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.

BcXyl, a ß-xylosidase Isolated from Brunfelsia Calycina Flowers with Anthocyanin-ß-glycosidase Activity.

Brunfelsia calycina flowers lose anthocyanins rapidly and are therefore well suited for the study of anthocyanin degradation mechanisms, which are unclear in planta. Here, we isolated an anthocyanin-ß-glycosidase from B. calycina petals. The MS/MS (Mass Spectrometry) peptide sequencing showed that the enzyme (72 kDa) was a ß-xylosidase (BcXyl). The enzyme showed high activity to p-Nitrophenyl-ß-d-galactopyranoside (pNPGa) and p-Nitrophenyl-ß-d-xylopyranoside (pNPX), while no activity to p-Nitrophenyl-ß-d-glucopyranoside (pNPG) or p-Nitrophenyl-ß-D-mannopyranoside (pNPM) was seen. The optimum temperature of BcXyl was 40 °C and the optimum pH was 5.0. The enzyme was strongly inhibited by 1 mM D-gluconate and Ag⁺. HPLC (High Performance Liquid Chromatography) analysis showed that BcXyl catalyzed the degradation of an anthocyanin component of B. calycina, and the release of xylose and galactose due to hydrolysis of glycosidic bonds by BcXyl was detected by GC (Gas Chromatography) /MS. A full-length mRNA sequence (2358 bp) of BcXyl (NCBI No. MK411219) was obtained and the deduced protein sequence shared conserved domains with two anthocyanin-ß-glycosidases (Bgln and BadGluc, characterized in fungi). BcXyl, Bgln and BadGluc belong to AB subfamily of Glycoside hydrolase family 3. Similar to BcPrx01, an anthocyanin-degradation-related Peroxidase (POD), BcXyl was dramatically activated at the stage at which the rapid anthocyanin degradation occurred. Taken together, we suggest that BcXyl may be the first anthocyanin-ß-glycosidase identified in higher plants.

Heterologous expression, purification and biochemical characterization of a new xylanase from Myceliophthora heterothallica F.2.1.4.

Myceliophthora heterothallica is a thermophilic fungus potentially relevant for the production of enzymes involved in the degradation of plant biomass. A xylanase encoding gene of this species was identified by means of RT-PCR using primers designed based on a xylanase coding sequence (GH11) of the fungus M. thermophila. The obtained gene was ligated to the vector pET28a(+) and the construct was transformed into Escherichia coli cells. The recombinant xylanase (r-ec-XylMh) was heterologously expressed, and the highest activity was observed at 55 °C and pH 6. The enzyme stability was greater than 70% between pH 4.5 and 9.5 and the inclusion of glycerol (50%) resulted in a significant increase in thermostability. Under these conditions, the enzyme retained more than 50% residual activity when incubated at 65 °C for 1 h, and approximately 30% activity when incubated at 70 °C for the same period. The tested cations did not increase xylanolytic activity, and the enzyme indicated significant tolerance to several phenolic compounds after 24 h, as well as high specificity for xylan, with no activity for other substrates such as CMC (carboxymethylcellulose), Avicel, pNPX (p-nitrophenyl-ß-D-xylopyranoside) and pNPA (p-nitrophenyl-α-L-arabinofuranoside), and is thus, of potential relevance in pulp bleaching.

Cloning, overexpression and characterization of a thermostable ß-xylosidase from Thermotoga petrophila and cooperated transformation of ginsenoside extract to ginsenoside 20(S)-Rg3 with a ß-glucosidase.

A thermostable ß-xylosidase gene Tpexyl3 from Thermotoga petrophila DSM 13,995 was cloned and overexpressed by Escherichia coli. Recombinant Tpexyl3 was purified, and its molecular weight was approximately 86.7 kDa. Its optimal activity was exhibited at pH 6.0 and 90 °C. It had broad specificity to xylopyranosyl, arabinopyranosyl, arabinofuranosyl and glucopyranosyl moieties. The ß-xylosidase activity of the recombinant Tpexyl3 was 6.81 U/mL in the LB medium and 151.4 U/mL in a 7.5 L bio-reactor. It was applied to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, which was combined with thermostable ß-glucosidase Tpebgl3. After transforming under optimal condition, the 20 g/L of ginsenoside extract was transformed into 6.28 g/L of Rg3 within 90 min, with a corresponding molar conversion of 95.0% and Rg3 productivity of 1793.49 mg/L/h, respectively. This study is the highest report of a GH3 family glycosidase with arabinopyranosidase activity and also the first report on the high substrate concentration bioconversion of ginsenoside extract to ginsenoside 20(S)-Rg3 by using two thermostable glycosidases.

Highly Efficient Biotransformation of Astragaloside IV to Cycloastragenol by Sugar-Stimulated ß-Glucosidase and ß-Xylosidase from Dictyoglomus thermophilum.

ß-Glucosidases and ß-xylosidases are two categories of enzymes that could cleave out nonreducing, terminal ß-D-glucosyl and ß-D-xylosyl residues with release of D-glucose and Dxylose, respectively. In this paper, two functional ß-glucosidase Dth3 and ß-xylosidase Xln-DT from Dictyoglomus thermophilum were heterologously expressed in E.coli BL21 (DE3). Dth3 and Xln-DT were relatively stable at 75°C and were tolerant or even stimulated by glucose and xylose. Dth3 was highly tolerant to glucose with a Ki value of approximately 3 M. Meanwhile, it was not affected by xylose in high concentration. The activity of Xln-DT was stimulated 2.13- fold by 1 M glucose and 1.29-fold by 0.3 M xylose, respectively. Furthermore, the ß- glucosidase Dth3 and ß-xylosidase Xln-DT showed excellent selectivity to cleave the outer C-6 and C-3 sugar moieties of ASI, which established an effective and green method to produce the more pharmacologically active CAG, an exclusive telomerase activator. We measured temperature, pH and dosage of enzyme using a single-factor experiment in ASI biotransformation. After optimization, the optimal reaction conditions were as follows: 75°C, pH 5.5, 1 U of Dth3 and 0.2 U of Xln-DT, respectively. Under the optimized conditions, 1 g/l ASI was transformed into 0.63 g/l CAG with a corresponding molar conversion of 94.5% within 3 h. This is the first report to use the purified thermostable and sugar-tolerant enzymes from Dictyoglomus thermophilum to hydrolyze ASI synergistically, which provides a specific, environment-friendly and cost-effective way to produce CAG.

Expression and characterisation of a pH and salt tolerant, thermostable and xylose tolerant recombinant GH43 ß-xylosidase from Thermobifida halotolerans YIM 90462T for promoting hemicellulose degradation.

A gene encoding a ß-xylosidase (designated as Thxyl43A) was cloned from strain Thermobifida halotolerans YIM 90462T. The open reading frame of this gene encodes 550 amino acid residues. The gene was over-expressed in Escherichia coli and the recombinant protein was purified. The monomeric Thxyl43A protein presented a molecular mass of 61.5 kDa. When p-nitrophenyl-ß-d-xylopyranoside was used as the substrate, recombinant Thxyl43A exhibited optimal activity at 55 °C and pH 4.0 to 7.0, being thermostable by maintaining 47% of its activity after 30 h incubation at 55 °C. The recombinant enzyme retained more than 80% residual activity after incubation at pH range of 4.0 to 12.0 for 24 h, respectively, which indicated notable thermostability and pH stability of Thxyl43A. Moreover, Thxyl43A displayed high catalytic activity (> 60%) in presence of 5-35% NaCl (w/v) or 1-20% ionic liquid (w/v) or 1-50 mM xylose. These properties suggest that Thxyl43A has potential for promoting hemicellulose degradation and other industrial applications.

Purification and Characterization of a recombinant ß-Xylosidase from Bacillus licheniformis ATCC 14580 into E. coli Bl21.

Present research work is aimed to purify and characterize a recombinant ß-xylosidase enzyme which was previously cloned from Bacillus licheniformis ATCC 14580 in to Escherichia coli BL21. Purification of recombinant enzyme was carried out by using ammonium sulphate precipitation method followed by single step immobilized metal ion affinity chromatography. Specific activity of purified recombinant ß-xylosidase enzyme was 20.78 Umg-1 with 2.58 purification fold and 33.75% recovery. SDS-PAGE was used to determine the molecular weight of recombinant purified ß-xylosidase and it was recorded as 52 kDa. Purified enzyme showed stability upto 90°C within a pH range of 3-8 with and optimal temperature and pH, 55ºC and 7.0, respectively. The enzyme activity was not considerably affected in the presence of EDTA. An increase in the enzyme activity was found in the manifestation of Mg+2. Enzyme activity was also increased by 6%, 18% and 22% in the presence of 1% Tween 80, ß-mercaptoethanol and DTT, respectively. Higher concentrations (10 - 40%) of organic solvents did not show any effect upon activity of enzyme. All these characteristics of the recombinant enzyme endorsed it as a potential candidate for biofuel industry.

New xylanolytic enzyme from Geobacillus galactosidasius BS61 from a geothermal resource in Turkey.

In this study, isolation, conventional and molecular characterizations of ten thermophilic bacteria from Rize/Ayder were carried out. Xylanase from Geobacillus galactosidasius BS61 (GenBank number: KX447660) was purified by acetone precipitation, Diethylaminoethyl-cellulose and Sephadex G-100 chromatographies. The xylanase of G. galactosidasius BS61 in clarifying fruit juice was also investigated. Enzyme was purified 29.80-fold with 75.18% yield; and molecular weight was determined as 78.15 kDa. The optimum temperature of xylanase was 60 °C. The enzyme activity was maintained fully after 24 h and over 50% after 168 h at pH 4.0-10.0, while optimum pH was 7.0. Km and Vmax for beech wood xylan were measured as 3.18 mg mL-1, 123 U mg protein-1. In addition, Ca2+, Na+, Al3+, Zn2+, Cd2+, Mg2+, Ni2+, Cu2+ had decreasing effect on enzyme activity, while enzyme activity had been protected against anions, especially HSO3- and HPO42- stimulated enzyme activity. Xylanase applications (with 15 U/mL enzyme activity) in orange and pomegranate juices were increased; and the sugar and turbidity amounts were reduced 17.36% ±â€¯1.18 and 30.52 ±â€¯1.23, respectively. These results indicated that the xylanase of G. galactosidasius BS61 has biotechnological potential in juice clarification due to its stability against metal ions, chemicals and high pH-values.

Purification and Characterisation of a Thermostable ß-Xylosidase from Aspergillus niger van Tieghem of Potential Application in Lignocellulosic Bioethanol Production.

A locally isolated strain of Aspergillus niger van Tieghem was found to produce thermostable ß-xylosidase activity. The enzyme was purified by cation and anion exchange and hydrophobic interaction chromatography. Maximum activity was observed at 70-75 °C and pH 4.5. The enzyme was found to be thermostable retaining 91 and 87% of its original activity after incubation for 72 h at 60 and 65 °C, respectively, with 52% residual activity detected after 18 h at 70 °C. Available data indicates that the purified ß-xylosidase is more thermostable over industrially relevant prolonged periods at high temperature than those reported from other A. niger strains. Maximum activity was observed on p-nitrophenyl-ß-D-xylopyranoside and the enzyme also hydrolysed p-nitrophenyl ß-D-glucopyranoside and p-nitrophenyl α-L-arabinofuranoside. The purified enzyme acted synergistically with A. niger endo-1,4-ß-xylanase in the hydrolysis of beechwood xylan at 65 °C. During hydrolysis of pretreated straw lignocellulose at 70 °C using a commercial lignocellulosic enzyme cocktail, inclusion of the purified enzyme resulted in a 19-fold increase in the amount of xylose produced after 6 h. The results observed indicate potential suitability for industrial application in the production of lignocellulosic bioethanol where thermostable ß-xylosidase activity is of growing interest to maximise the enzymatic hydrolysis of lignocellulose.

Enhanced Purification Efficiency and Thermal Tolerance of Thermoanaerobacterium aotearoense ß-Xylosidase through Aggregation Triggered by Short Peptides.

To simplify purification and improve heat tolerance of a thermostable ß-xylosidase (ThXylC), a short ELK16 peptide was attached to its C-terminus, which is designated as ThXylC-ELK. Wild-type ThXylC was normally expressed in soluble form. However, ThXylC-ELK assembled into aggregates with 98.6% of total ß-xylosidase activity. After simple centrifugation and buffer washing, the ThXylC-ELK particles were collected with 92.57% activity recovery and 95% purity, respectively. Meanwhile, the wild-type ThXylC recovery yield was less than 55% after heat inactivation, affinity and desalting chromatography followed by HRV 3C protease cleavage purification. Catalytic efficiency ( Kcat/ Km) was increased from 21.31 mM-1 s-1 for ThXylC to 32.19 mM-1 s-1 for ThXylC-ELK accompanied by a small increase in Km value. Heat tolerance of ThXylC-ELK at high temperatures was also increased. The ELK16 peptide attachment resulted in 6.2-fold increase of half-life at 65 °C. Released reducing sugars were raised 1.3-fold during sugar cane bagasse hydrolysis when ThXylC-ELK was supplemented into the combination of XynAΔSLH and Cellic CTec2.

A halotolerant bifunctional ß-xylosidase/α-l-arabinofuranosidase from Colletotrichum graminicola: Purification and biochemical characterization.

A ß-xylosidase from Colletotrichum graminicola (Bxcg) was purified. The enzyme showed high halotolerance, retaining about 63% of the control activity in the presence of 2.5molL-1 NaCl. The presence of NaCl has not affected the optimum reaction temperature (65°C), but the optimum pH was slightly altered (from 4.5 to 5.0) at high salt concentrations. Bxcg was fully stable at 50°C for 24h and over a wide pH range even in the presence of NaCl. In the absence of salt Bxcg hydrolyzed p-nitrophenyl-ß-d-xylopyranoside with maximum velocity of 348.8±11.5Umg-1 and high catalytic efficiency (1432.7±47.3Lmmol-1s-1). Bxcg revealed to be a bifunctional enzyme with both ß-xylosidase and α-l-arabinofuranosidase activities, and hydrolyzed xylooligosaccharides containing up to six pentose residues. The enzyme showed high synergistic effect (3.1-fold) with an endo-xylanase for the hydrolysis of beechwood xylan, either in the absence or presence of 0.5molL-1 NaCl, and was tolerant to different organic solvents and surfactants. This is the first report of a halotolerant bifunctional ß-xylosidase/α-l-arabinofuranosidase from C. graminicola, and the enzyme showed attractive properties for application in lignocellulose hydrolysis, particularly under high salinity and/or in the presence of residues of pretreatment steps.

GH11 xylanase from Aspergillus tamarii Kita: Purification by one-step chromatography and xylooligosaccharides hydrolysis monitored in real-time by mass spectrometry.

The present study describes the one-step purification and biochemical characterization of an endo-1,4-ß-xylanase from Aspergillus tamarii Kita. Extracellular xylanase was purified to homogeneity 7.43-fold through CM-cellulose. Enzyme molecular weight and pI were estimated to be 19.5kDa and 8.5, respectively. The highest activity of the xylanase was obtained at 60°C and it was active over a broad pH range (4.0-9.0), with maximal activity at pH 5.5. The enzyme was thermostable at 50°C, retaining more than 70% of its initial activity for 480min. The K0.5 and Vmax values on beechwood xylan were 8.13mg/mL and 1,330.20µmol/min/mg of protein, respectively. The ions Ba2+ and Ni2+, and the compounds ß-mercaptoethanol and DTT enhanced xylanase activity, while the heavy metals (Co2+, Cu2+, Hg+, Pb2+ and Zn2+) strongly inhibited the enzyme, at 5mM. Enzymatic hydrolysis of xylooligosaccharides monitored in real-time by mass spectrometer showed that the shortest xylooligosaccharide more efficiently hydrolyzed by A. tamarii Kita xylanase corresponded to xylopentaose. In agreement, HPLC analyzes did not detect xylopentaose among the hydrolysis products of xylan. Therefore, this novel GH11 endo-xylanase displays a series of physicochemical properties favorable to its application in the food, feed, pharmaceutical and paper industries.

Purification and characterization of novel bi-functional GH3 family ß-xylosidase/ß-glucosidase from Aspergillus niger ADH-11.

ß-Xylosidase plays an important role in xylan degradation by relieving the end product inhibition of endo-xylanase caused by xylo-oligosaccharides. ß-Xylosidase has a wide range of applications in food, feed, paper and pulp, pharmaceutical industries and in bioconversion of lignocellulosic biomass. Hence, in the present study focused on purification, biochemical characterization and partial sequencing of purified ß-xylosidase from xylanolytic strain Aspergillus niger ADH-11. Acetone precipitation followed by GPC using Sephacryl S-200 yielded 20.59-fold purified ß-xylosidase with 58.30% recovery. SDS-PAGE analysis of purified ß-xylosidase relieved a monomeric subunit with a molecular weight 120.48kDa. Kinetic parameters of purified ß-xylosidase viz Km, Vmax, Kcat and catalytic efficiency were assessed. Purified ß-xylosidase was additionally active on p-nitrophenyl-ß-d-glucopyranoside substrate also. Moreover, peptide mass fingerprinting analysis support our biochemical studies and showed that the purified protein is a novel ß-xylosidase with ß-glucosidase activity and belongs to the bi-functional GH3 superfamily. Besides, tolerance of purified ß-xylosidase towards glucose and xylose was also assessed.

Expression of catalytically efficient xylanases from thermophilic fungus Malbranchea cinnamomea for synergistically enhancing hydrolysis of lignocellulosics.

In this study, two xylanase genes (GH10 and GH11) derived from Malbranchea cinnamomea, designated as XYN10A_MALCI and XYN11A_MALCI, respectively, were expressed in Pichia pastoris X33. The maximum level of xylanase expression was found to be 24.3U/ml for rXYN10A_MALCI and 573.32U/ml for rXYN11A_MALCI. The purified recombinant rXYN11A_MALCI was stable at 70°C and catalytically active against a variety of substituted (arabinoxylans) as well as unsubstituted xylans. The hydrolytic potential of recombinant xylanases for enhancing the hydrolysis of acid/alkali pretreated lignocellulosics (rice straw and bagasse) by the commercial cellulase Cellic CTec2 was assessed which revealed that both rXYN10A_MALCI and rXYN11A_MALCI act synergistically with commercial cellulases and resulted in 1.54 and 1.58 folds improved hydrolysis of acid treated rice straw and alkali treated rice straw using cocktail comprising of Cellic CTec2 and XYN11A_MALCI (8:2 ratio) when compared to Cellic CTec2 alone at same protein loading rate of (∼5.7mg/g biomass).

A bifunctional cellulase-xylanase of a new Chryseobacterium strain isolated from the dung of a straw-fed cattle.

A new cellulolytic strain of Chryseobacterium genus was screened from the dung of a cattle fed with cereal straw. A putative cellulase gene (cbGH5) belonging to glycoside hydrolase family 5 subfamily 46 (GH5_46) was identified and cloned by degenerate PCR plus genome walking. The CbGH5 protein was overexpressed in Pichia pastoris, purified and characterized. It is the first bifunctional cellulase-xylanase reported in GH5_46 as well as in Chryseobacterium genus. The enzyme showed an endoglucanase activity on carboxymethylcellulose of 3237 µmol min-1  mg-1 at pH 9, 90 °C and a xylanase activity on birchwood xylan of 1793 µmol min-1  mg-1 at pH 8, 90 °C. The activity level and thermophilicity are in the front rank of all the known cellulases and xylanases. Core hydrophobicity had a positive effect on the thermophilicity of this enzyme. When similar quantity of enzymatic activity units was applied on the straws of wheat, rice, corn and oilseed rape, CbGH5 could obtain 3.5-5.0× glucose and 1.2-1.8× xylose than a mixed commercial cellulase plus xylanase of Novozymes. When applied on spent mushroom substrates made from the four straws, CbGH5 could obtain 9.2-15.7× glucose and 3.5-4.3× xylose than the mixed Novozymes cellulase+xylanase. The results suggest that CbGH5 could be a promising candidate for industrial lignocellulosic biomass conversion.